GFP-OPTN 217–577 –induced pexophagy in HEK-293 cells depends on endogenous OPTN. Flp-In T-REx 293 cells, stably expressing po-mKeima, were seeded in 6-well plates and transfected with either control (CT) or OPTN-targeting DsiRNA. On day 2, cells were transfected with plasmids encoding either GFP- or GFP-OPTN217–577. On day 3, DsiRNA transfections were repeated. On day 4, cells were harvested and subjected to either FACS analysis or IB using antibodies against the indicated proteins. Pexophagy was assessed by gating single-cell GFP-positive populations for a decrease in fluorescence intensity in the neutral channel. (A) Representative immunoblots confirming the efficacy of OPTN DsiRNA knockdown. Arrows indicate specific protein bands. (B) Densitometric quantification of relative normalized amounts (R-NA) of OPTN in C (n = 3). Bars represent mean ± SD of three biological replicates (****, P < 0.0001). (C) Comparison of the electrophoretic mobility of endogenous OPTN (CT DsiRNA condition) and GFP-OPTN217–577 (OPTN DsiRNA/GFP-OPTN217–577 condition). Molecular mass markers (in kDa) are indicated on the left. (D) Representative flow cytometry plots from each group (n = 3). Colors indicate cell density at a given position. (E) Quantification of the percentage of cells within the gated population. Data are presented as mean ± SD from three independent biological replicates. Statistical comparisons reflect the effects of OPTN downregulation and GFP-OPTN217–577 overexpression (ns, nonsignificant; *, P < 0.05; **, P < 0.01;****, P < 0.0001). Source data are available for this figure: SourceData F6.